Methylmalonate-semialdehyde dehydrogenase is induced in auxin-stimulated and zinc-stimulated root formation in rice

Proteins induced in rice by auxin and zinc were determined by proteome analysis. Cultured suspension cells of rice were treated with 2,4-dichlorophenoxyacetic acid and ZnSO₄ and then proteins were separated by two-dimensional polyacrylamide gel electrophoresis; seven proteins were found to be induced by auxin and zinc. Of these seven, methylmalonate-semialdehyde dehydrogenase (MMSDH) was elevated by treatment with auxin alone. MMSDH was detected in cultured suspension cells, root and leaf sheath, but not in leaf blades. MMSDH responded to auxin and gibberellin, but did not respond to brassinolide and cytokinin. Furthermore, the amount of MMSDH in slr1, a constitutive gibberellin response mutant, was 2-fold that of wild type. MMSDH mRNA and protein were stimulated in root formation induced by auxin and/or zinc over a 4-week period. These results suggest that MMSDH may be necessary for root formation in rice induced by auxin and/or zinc.Abbreviations BA 6-Benzylaminopurine - BL Brassinolide - CBB Coomassie Brilliant Blue - 2,4-D 2,4-Dichlorophenoxyacetic acid - GA Gibberellic acid - IAA Indole-3-acetic acid - MALDI-TOF MS Matrix-assisted laser desorption-ionization time-of-flight mass spectrometry - MMSDH Methylmalonate-semialdehyde dehydrogenase - 2D-PAGE Two-dimensional polyacrylamide gel electrophoresis - PVDF Polyvinylidene difluoride     

Characterization of a histidine- and alanine-rich protein showing interaction with calreticulin in rice

Calreticulin (CRT) is a major calcium-sequestering protein in the endoplasmic reticulum and has been implicated in a variety of cellular functions. To analyze the function of CRT in rice, a yeast two-hybrid protein interaction assay was used for identifying interacting proteins. Fourteen of 17 interacting cDNA clones found coded for a novel histidine- and alanine-rich protein (OsHARP) of 342 amino acid residues. The mRNA expression level of OsHARP was up-regulated in rice seedlings treated with gibberellin (GA), but not ABA and showed a similar pattern as OsCRT mRNA. Rice plants transformed with the OsHARP promoter-GUS construct showed GUS staining in the basal parts of leaf sheaths, and although GUS activity increased when treated with GA₃, it was not as high an increase as when mRNA was analyzed. To elucidate the role of OsHARP in leaf sheath elongation, antisense OsHARP transgenic rice lines were constructed. The antisense OsHARP transgenic rice plants were consistently shorter than the vector control under normal conditions. To examine whether OsHARP expression would affect other proteins, basal leaf sheaths from antisense OsHARP transgenic rice plants were analyzed using proteomic techniques. In antisense transgenic-rice OsHARP plants, OsCRT was down-regulated and the levels of 20 other proteins were changed compared to the pattern of the vector control. These results signify an important role of HARP in rice leaf sheath cell division or elongation and suggest that CRT may interact with HARP during certain stages of development.     

Cooperative action of SLR1 and SLR2 is required for lateral root-specific cell elongation in maize

Lateral roots play an important role in water and nutrient uptake largely by increasing the root surface area. In an effort to characterize lateral root development in maize (Zea mays), we have isolated from Mutator (Mu) transposon stocks and characterized two nonallelic monogenic recessive mutants: slr1 and slr2 (short lateral roots1 and 2), which display short lateral roots as a result of impaired root cell elongation. The defects in both mutants act specifically during early postembryonic root development, affecting only the lateral roots emerging from the embryonic primary and seminal roots but not from the postembryonic nodal roots. These mutations have no major influence on the aboveground performance of the affected plants. The double mutant slr1; slr2 displays a strikingly different phenotype than the single mutants. The defect in slr1; slr2 does not only influence lateral root specific cell elongation, but also leads to disarranged cellular patterns in the primary and seminal roots. However, the phase-specific nature of the single mutants is retained in the double mutant, indicating that the two loci cooperate in the wild type to maintain the lateral root specificity during a short time of early root development.     

Overexpression of a GRAS protein lacking the DELLA domain confers altered gibberellin responses in rice

The rice SLR1 (SLENDER RICE 1) gene encodes a DELLA protein that belongs to a subfamily of the GRAS protein superfamily and that functions as a repressor of gibberellin (GA) signaling. Based on the constitutive GA response phenotype of slr1 mutants, SLR1 has been thought to be the sole DELLA-type protein suppressing GA signals in rice. However, in rice genome databases we identified two sequences homologous to SLR1: SLR1-like1 and -2 (SLRL1 and -2). SLRL1 and SLRL2 contain regions with high similarity to the C-terminal conserved domains in SLR1, but lack the N-terminal conserved region of the DELLA proteins. The expression of SLRL1 was positively regulated by GA at the mRNA level and occurred preferentially in reproductive organs, whereas SLRL2 was moderately expressed in mature leaf organs and was not affected by GA. Transformation of SLRL1 into the slr1 mutant rescued the slender phenotype of this mutant. Moreover, overexpression of SLRL1 in normal rice plants induced a dwarf phenotype with an increased level of OsGA20ox2 gene expression and diminished the GA-induced shoot elongation, suggesting that SLRL1 acts as a repressor of GA signaling. Consistent with the fact that SLRL1 does not have a DELLA domain, which is essential for degradation of DELLA proteins, a level of SLRL1 protein was not degraded by application of gibberellic acid. However, the repressive activity of SLRL1 against GA signaling was much weaker than a truncated SLR1 lacking the DELLA domain. Based on these characteristics of SLRL1, the functional roles of SLRL1 in GA signaling in rice are discussed.     

Altered life cycle in Arabidopsis plants expressing PsUGT1, a UDP-glucuronosyltransferase-encoding gene from pea

Alfalfa (Medicago sativa) and Arabidopsis were used as model systems to examine molecular mechanisms underlying developmental effects of a microsomal UDP-glucuronosyltransferase-encoding gene from pea (Pisum sativum; PsUGT1). Alfalfa expressing PsUGT1 antisense mRNA under the control of the cauliflower mosaic virus (CaMV) 35S promoter exhibited delayed root emergence, reduced root growth, and increased lateral root development. The timing of root emergence in wild-type and antisense plants was correlated with the transient accumulation of auxin at the site of root emergence. Cell suspension cultures derived from the antisense alfalfa plants exhibited a delay in cell cycle from 24-h in the wild-type plants to 48-h in the antisense plants. PsUGT1::uidA was introduced into Arabidopsis to demonstrate that, as in alfalfa and pea, PsUGT1 expression occurs in regions of active cell division. This includes the root cap and root apical meristems, leaf primordia, tips of older leaves, and the transition zone between the hypocotyl and the root. Expression of PsUGT1::uidA colocalized with the expression of the auxin-responding reporter DR5::uidA. Co-expression of DR5::uidA in transgenic Arabidopsis lines expressing CaMV35S::PsUGT1 revealed that ectopic expression of CaMV35S::PsUGT1 is correlated with a change in endogenous auxin gradients in roots. Roots of ecotype Columbia expressing CaMV35S::PsUGT1 exhibited distinctive responses to exogenous naphthalene acetic acid. Completion of the life cycle occurred in 4 to 6 weeks compared with 6 to 7 weeks for wild-type Columbia. Inhibition of endogenous ethylene did not correct this early senescence phenotype.     

Promotion of leaf sheath growth by gibberellic acid in a dwarf mutant of rice

The mechanism of gibberellin (GA)-induced leaf sheath growth was examined using a dwarf mutant of rice (Oryza sativa L. cv. Tan-ginbozu) treated in advance with an inhibitor of GA biosynthesis. Gibberellic acid (GA₃) enhanced the growth of the second leaf sheath, but auxins did not. Measurement of the mitotic index and cell size revealed that cell elongation rather than cell division is promoted by GA₃. Gibberellic acid increased the extensibility of cell walls in the elongation zone of the leaf sheath. It also increased the total amount of osmotic solutes including sugars in the leaf sheath, but did not increase the osmotic concentration of the cell sap, due to an accompanying increase in cell volume by water absorption. In the later stage of GA₃-induced growth, starch granules completely disappeared from leaf sheath cells, whereas dense granules remained in control plants. These findings indicate that GA enhances cell elongation by increasing wall extensibility, osmotic concentration being kept unchanged by starch degradation. Received: 28 August 1997 / Accepted: 16 October 1997     

Functional analysis of OsTUB8, an anther-specific β-tubulin in rice

Microtubules play important roles in many cellular processes, such as cell division and cell elongation in plants. β-tubulins (TUB), which are one of the basic components of microtubules, are encoded by multigene family in eukaryotes and their nucleotide sequences are highly conserved in protein coding regions. OsTUB8 that was expressed in rice anthers was characterized with a multi-level approach. At the protein level, OsTUB8 was expressed mainly in anthers compared to callus, root, leaf sheath and leaf blade. In situ hybridization and GUS fusion analysis revealed that OsTUB8 was expressed in vascular bundles of anther filaments and in pollen. OsTUB8 expression was lower in the anthers of GA-deficient mutants, ‘Tanginbozu’ and ‘Akibrarewaisei’, compared to those of their respective wild types. Transgenic rice expressing OsTUB8 in an antisense orientation were suppressed in the amount of seed set upon maturity. Antisense-transgenic rice plants were 20–60% shorter compared to the vector-only control. These results suggest that OsTUB8 might be differentially expressed in rice anthers due to the action of GA, and involved in the processes of vegetative growth and seed set in rice.     

Roles of OsCKI1, a rice casein kinase I, in root development and plant hormone sensitivity

Casein kinases are critical in cell division and differentiation across species. A rice cDNA fragment encoding a putative casein kinase I (CKI) was identified via cDNA macroarray under brassinosteroid (BR) treatment, and a 1939-bp full-length cDNA, OsCKI1, was isolated and found to encode a putative 463-aa protein. RT-PCR and Northern blot analysis indicated that OsCKI1 was constitutively expressed in various rice tissues and upregulated by treatments with BR and abscisic acid (ABA). Enzymatic assay of recombinant OsCKI1 proteins expressed in Escherichia coli showed that the protein was capable of phosphorylating casein. The physiological roles of OsCKI1 were studied through antisense transgenic approaches, and homozygous transgenic plants showed abnormal root development, including fewer lateral and adventitious roots, and shortened primary roots as a result of reduced cell elongation. Treatment of wild-type plants with CKI-7, a specific inhibitor of CKI, also confirmed these functions of OsCKI1. Interestingly, in transgenic and CKI-7-treated plants, exogenously supplied IAA could restore normal root development, and measurement of free IAA content in CKI-deficient primary and adventitious roots revealed altered auxin content, indicating that OsCKI1 is involved in auxin metabolism or that it may affect auxin levels. Transgenic plants were less sensitive than control plants to ABA or BR treatment during germination, suggesting that OsCKI1 may be involved in various hormone-signaling pathways. OsCKI1-GFP fusion studies revealed the localization of OsCKI1 to the nucleus, suggesting a possible involvement in regulation of gene expression. In OsCKI1-deficient plants, differential gene expression was investigated using cDNA chip technology, and results indicated that genes related to signal transduction and hormone metabolism were indeed with altered expression.     

Elongation rates and endogenous indoleacetic acid levels in roots of pea mutants differing in internode length

Growth of the primary root of 12 genotypes of peas ( Pisum sativum ) differing in their stem height was recorded for 14 days. The growth rate of roots of wild-type tall, gibberellin (GA)-deficient le dwarf or slender mutants (with la cry^s ) was similar (3 cm day⁻¹); that of severely GA-deficient nana ( na-1 ) plants was 50% of wild-type but elongation ceased after 8 days; moderately severe dwarf GA-deficient lines ls-1 and lh-1 had a 15% reduction in elongation rate but displayed no time-dependent slowing of the growth rate and brassinosteroid-insensitive and -deficient dwarfs lka and lkb showed slightly decreased root elongation. GA (levels reported in Yaxley et al. 2001 ) is not substantially limiting to root growth until it is severely deficient. The terminal 3 cm of roots of tall plants contained about 25 or 35 ng g⁻¹ fresh weight indole-3-acetic acid (IAA), depending on the genetic background, and le-1 dwarfs were similar. Nana ( na-1 ) had less than 50% the level of IAA of tall, all the moderately severe dwarfs had reductions of about 30% and the slender plants had about 40% more IAA than the corresponding wild-type. With the exception of slender plants, IAA level in the root tips correlated with root elongation. Root growth seems to be promoted by IAA within the range of the internal concentrations detected. Nana plants had a reduced amount of IAA and a lower root-growth rate. Whereas external application of IAA always inhibits root growth, even at very low concentrations, root growth is not similarly inhibited by internal IAA as slender plants had the highest IAA level and growth rate similar to wild-type, regardless of the shoot GA content.     

WEG1, which encodes a cell wall hydroxyproline-rich glycoprotein,is essential for parental root elongation controlling lateral root formation in rice

Lateral roots (LRs) determine the overall root system architecture, thus enabling plants to efficiently explore their underground environment for water and nutrients. However, the mechanisms regulating LR development are poorly understood in monocotyledonous plants. We characterized a rice mutant, wavy root elongation growth 1 (weg1), that produced higher number of long and thick LRs (L-type LRs) formed from the curvatures of its wavy parental roots caused by asymmetric cell growth in the elongation zone. Consistent with this phenotype, was the expression of the WEG1 gene, which encodes a putative member of the hydroxyproline-rich glycoprotein family that regulates cell wall extensibility, in the root elongation zone. The asymmetric elongation growth in roots is well known to be regulated by auxin, but we found that the distribution of auxin at the apical region of the mutant and the wild-type roots was symmetric suggesting that the wavy root phenotype in rice is independent of auxin. However, the accumulation of auxin at the convex side of the curvatures, the site of L-type LR formation, suggested that auxin likely induced the formation of L-type LRs. This was supported by the need of a high amount of exogenous auxin to induce the formation of L-type LRs. These results suggest that the MNU-induced weg1 mutated gene regulates the auxin-independent parental root elongation that controls the number of likely auxin-induced L-type LRs, thus reflecting its importance in improving rice root architecture.     

A novel brassinolide-enhanced gene identified by cDNA microarray is involved in the growth of rice

Brassinosteroids (BRs) are growth-promoting natural substances required for normal plant growth and development. To understand the molecular mechanism of BR action, a cDNA microarray containing 1265 rice genes was analyzed for expression differences in rice lamina joint treated with brassinolide (BL). A novel BL-enhanced gene, designated OsBLE2, was identified and cloned. The full-length cDNA is 3243 bp long, encoding a predicted polypeptide of 761 amino acid residues and nine possible transmembrane regions. OsBLE2 expression was most responsive to BL in the lamina joint and leaf sheath in rice seedlings. Besides, auxin and gibberellins also increased its expression. OsBLE2 expressed more, as revealed by in situ hybridization, in vascular bundles and root primordia, where the cells are actively undergoing division, elongation, and differentiation. Transgenic rice expressing antisense OsBLE2 exhibits various degrees of repressed growth. BL could not enhance its expression in transgenic rice expressing antisense BRI1, a BR receptor, indicating that BR signaling to the enhanced expression of OsBLE2 is through BRI1. BL effect in the d1 mutant rice was much weaker than that in its wild-type control, indicating that heterotrimeric G protein may be a component of BRs signaling. These results suggest that OsBLE2 is involved in BL-regulated growth and development processes in rice.     

Identification and characterization of a gibberellin-regulated protein,which is ASR5, in the basal region of rice leaf sheaths

Gibberellins (GAs) regulate growth and development in higher plants. To identify GA-regulated proteins during rice leaf sheath elongation, a proteomic approach was used. Proteins from the basal region of leaf sheath in rice seedling treated with GA₃ were analyzed by fluorescence two-dimensional difference gel electrophoresis. The levels of abscisic acid-stress-ripening-inducible 5 protein (ASR5), elongation factor-1 beta, translationally controlled tumor protein, fructose-bisphosphate aldolase and a novel protein increased; whereas the level of RuBisCO subunit binding-protein decreased by GA₃ treatment. ASR5 out of these six proteins was significantly regulated by GA₃ at the protein level but not at the mRNA level in the basal region of leaf sheaths. Since this protein is regulated not only by abscisic acid but also by GA₃, these results indicate that ASR5 might be involved in plant growth in addition to stress in the basal regions of leaf sheaths.     

The slender rice mutant,with constitutively activated gibberellin signal transduction,has enhanced capacity for abscisic acid level

The slender rice (slr1-1) mutant, carrying a lethal and recessive single mutation, has a constitutive gibberellin (GA)-response phenotype and behaves as if it were saturated with GAs [Ikeda et al. (2001) Plant Cell 13, 999]. The SLR1 gene, with sequence homology to members of the plant-specific GRAS gene family, is a mediator of the GA signal transduction process. In the slender rice, GA-inducible alpha-amylase was produced from the aleurone layer without applying GA. GA-independent alpha-amylase production in the mutant was inhibited by applying abscisic acid (ABA). Shoot elongation in the mutant was also suppressed by ABA, indicating that the slender rice responds normally to ABA. Interestingly, shoot ABA content was 10-fold higher in the mutant than in the wild type, while there was no difference in root ABA content. Expression of the Rab16A gene, which is known to be ABA inducible, was about 10-fold higher in shoots of the mutant than in those of the wild type. These results indicate that constitutive activation of the GA signal transduction pathway by the slr1-1 mutation promotes the endogenous ABA level.